Nonclassical mechanisms to irreversibly suppress β-hematin crystal growth

Hematin crystallization is an essential element of heme detoxification of malaria parasites and its inhibition by antimalarial drugs is a common treatment avenue. We demonstrate at biomimetic conditions in vitro irreversible inhibition of hematin crystal growth due to distinct cooperative mechanisms that activate at high crystallization driving forces. The evolution of crystal shape after limited-time exposure to both artemisinin metabolites and quinoline-class antimalarials indicates that crystal growth remains suppressed after the artemisinin metabolites and the drugs are purged from the solution. Treating malaria parasites with the same agents reveals that three- and six-hour inhibitor pulses inhibit parasite growth with efficacy comparable to that of inhibitor exposure during the entire parasite lifetime. Time-resolved in situ atomic force microscopy (AFM), complemented by light scattering, reveals two molecular-level mechanisms of inhibitor action that prevent β-hematin growth recovery. Hematin adducts of artemisinins incite copious nucleation of nonextendable nanocrystals, which incorporate into larger growing crystals, whereas pyronaridine, a quinoline-class drug, promotes step bunches, which evolve to engender abundant dislocations. Both incorporated crystals and dislocations are known to induce lattice strain, which persists and permanently impedes crystal growth. Nucleation, step bunching, and other cooperative behaviors can be amplified or curtailed as means to control crystal sizes, size distributions, aspect ratios, and other properties essential for numerous fields that rely on crystalline materials.

called hemozoin that are formed within growing malarial parasites) and monitor growth of those seed crystals in vitro for 3 vs 13 days (or 3 days followed by 10 days more) via atomic force microscopy in the presence vs absence of quinoline antimalarial drugs and artemisinin -heme adducts. They interpret their data in terms of two modes of crystal growth inhibition, either reversible or irreversible. The topic is quite important and the team of authors are prominent and highly skilled researchers, however, some points need to be addressed as described below.
Major issues: 1) The key data are presented in Fig. 1, and distinguishing whether blue and orange bars in Fig. 1e are the same or different in height is essential to interpretation, however, no statistics are given (also bars in extended data Fig. 2). From the error bars (extended Fig. 2 has no error bars at all) it is difficult to ascertain which blue and orange bars are statistically the same vs different. Routine T test and recitation of calculated p values is sorely needed.
2) The authors propose that inhibition of crystal growth is irreversible if two criteria are met, the first being less crystal growth vs control (which is no inhibitor for 13 days) in the 10 days after inhibitor is removed following a 3 day incubation with inhibitor (criterion 1) and, second, that growth in the constant presence of inhibitor for 13 days is the same as growth following 3 days + followed by 10 days -inhibitor. Only 1 inhibitor (H-ARS) seems to satisfy both, but the title and text of the paper seems to imply that the inhibitors are irreversible, and Fig. 2 presents detailed arguments and a cartoon entitled "irreversible inhibition ... by H-ART" when the data in Fig. 1 show that H-ART satisfies neither criterion. The separation of crystal width vs length effects as reversible or irreversible in the text further confuses interpretation, are the authors implying that some inhibitors are reversible in one dimension but irreversible in another ? Some clarification in simple language is needed to assist the reader.
3) The crystal inhibition assays are done over 13 days in vitro under highly non physiologic conditions, but the authors imply that by comparing growth effects of live parasites vs the same inhibitors that somehow the measured crystal inhibition characteristics are relevant to understanding the mechanism of drug inhibition of hemozoin in vivo, in which hemozoin crystals are formed within hours. The highly artificial nature of the crystal formation measurements needs to be emphasized, with limitations on interpretation then highlighted.
Additional points: 4) pg. 1 "inevitably predicts" but then the next sentence seems to contradict this. 5) next line, "irreversible inhibition of hematin crystallization" ... at best, "in vitro under non physiological conditions". 6) next line following, "cooperative", how so ? This term has a formal definition in biological sciences, it is not clear how cooperativity in inhibition of crystal growth by any inhibitor studied is being ascertained or quantified. 7) bottom of pg 1, top pg 2 is very misleading, H-ART and H-ARS used in the paper are not drugs, they are drug heme adducts. ART drugs cannot be "purged" from the solution as implied, they become covalently attached to their intracellular targets. 8) 5 lines following, "copious nucleation" is not defined or quantified, what is meant by this phrase ? 9) pg 2 par 2 last line, reference 23 does not suggest "adducts ... form in the ... digestive vacuole" as implied, this paper uses NMR methods to assign meso carbon covalent attachment sites for ARTheme adducts formed in vitro. Heme detoxification suppression still stands as a pivotal treatment for malaria. Basic science towards understanding the nucleation of β-hematin crystals in the growth/biogenic medium and how antimalarial drugs can impair it is of importance. The manuscript characterizes the β-hematin growth under antimalarial drugs (old and new ones) towards advancing the underlying mechanisms of inhibition. A key advance here is to address each contribution of reversible and irreversible inhibitory steps. Notably, authors have identified irreversible inhibition of β-hematin crystallization by some drugs, and importantly, aimed to understand the underlying reason for the phenomena and to correlate this with the antiplasmodium activity. Overall, though there are many questions remaining about how exactly these compounds exert heme toxification and antiparasitic effects, there is sufficient new insights to support its publication in Communications Biology. That said, there are some important concerns outlined below should be addressed.
A) The notion that in low concentration, hematin-artemisinin adducts (H-ARS/H-ART) did not efficiently inhibit the growth of young parasites (early or late rings) is a substantial phenotype shift and a novelty in comparison to the parental Artemisinin/Artesunate efficacy. Presumably, adducts are devoid in peroxide bond necessary to alkylate protein; however, adducts were previously able, at least in a high concentration (500 nM), to kill early rings in the RSA (DOI 10.1074/jbc.RA120.016115). To reconciliate this apparent paradox, authors are encouraged to discuss this or experimentally address the parasite survival in Figure 5 panels B and C using high concentrations of H-ARS/H-ART (up to 1 microM).
B) Yet regarding the drug concentration, the authors report IC50 values of 6 h expose versus 72 h (no wash out). The determination of IC50s 6h was presumably intended to reflect the irreversibility of parasite inhibition and to precisely correlate this phenomenon with the drug interaction with hemozoin crystals; this is a novelty. A cut-off of IC50 6 h > 12 folds the IC50 72 h was wisely established. Troublingly, the curves of IC50 values of 6 h expose for early and later ring do not seems like a sigmoid curve. The accuracy of regression-derived values is not clear either. In other words, why authors did not test compounds in higher concentration up to 1 microM in order to generate suitable sigmoid curves? C) In parts, a great novelty of the present study is the determination of phenotype signature of hematin-artemisinin adducts (H-ARS/H-ART). Their antiplasmodium activity are quite appealing. IC50 values determined within 72 h of continuous drug incubation indicate they are almost equipotent, this is consistent to the structure-activity relationship. That said, there are dissimilarities in the IC50 6-h that should be addressed/discussed. There are a couple concerns with this experiment, though. First, H-ART seems to kill trophozoites more efficiently than H-ARS. Even in higher concentration, there are still parasites surviving at H-ARS treatment. Subsequently, trophozoite survival fractions for H-ART is of 5, while of 11 for H-ARS. Does this behave in the same way for IC50 3-h (Extended Data Fig. 5 not depicted for H-ARS)? Conversely, we know that iron protoporphyrin IX (Fe-PPIX) can adsorb in wire glass and the plastic surface of a microplate. No evidence is provided to indicate that the H-ARS/H-ART can be truly washed out by the protocol used. Therefore, it is possible that parasites continue to be effectively exposed to the hematin-artemisinin adducts following the washout step, especially if no plate transfer was performed (see plate transfer, DOI: 10.1128/AAC.00574-16). This could be the reason for the dissimilarity in IC50 6h values. This could be a useful feature in therapeutic applications, but confounds interpretation of phenotype response. Fig. 6, all drugs except for mefloquine were quite consistent to the literature. For mefloquine, the IC50 of 88 nM is higher than typically observed in most the literature. Mefloquine supplied by Sigma-Aldrich (M2319) is provided as a partially DMSO-insoluble salt. Could this be the reason for the limited potency of mefloquine?

D) In a close inspection of the IC50 values from Extended Data
E) Yet regarding mefloquine. The irreversibility of parasite inhibition and the correlation with the drug interaction with hemozoin crystals (reversible/irreversible) is an important issue. For sure, all drugs tested here apart mefloquine (chloroquine, pyronaridine, and hematin-adducts) are of fast-action antimalarial activity (i.e., to decrease parasite viability over 24 h drug expose). Presumably, a fastaction property may correlate with the ability of a drug in inhibiting hemozoin crystal. It is largely assumed that heme augmentation can cause a fast-acting lethal event for the parasite cells (advocated by findings of Timothy Egan and Paul D. Roepe). However, mefloquine is not a fast-acting drug, rather than, it is a relatively slow (slower than CQ, faster than atovaquone). Authors are encouraged to discuss that for mefloquine, most precedent literature of phenotype activity (10.1038/nmicrobiol.2017.31; 10.1126/scitranslmed.aau3174; 10.1021/acs.accounts.1c00154) point out that other mechanisms are operative rather than hemozoin blockage alone. Perhaps, mefloquine ability to inhibit hemozoin crystals is a secondary mechanism. F) In the experimental design in Figure 5, it is not clear if a drug expose of 3 h was performed as denoted (3 or 6 hours). Indeed, a 3h data is only displayed in the supporting information. Otherwise, just kept 6 h in panel A. G) In Panel E, of Figure 1, it was not clear in the "no drug" group what is the difference between the two columns? Is it 3-days versus 13 days? H) Authors are encouraged to display, either in the main text or in the supporting information, a table with the full set of IC50 values and standard deviation, in addition to the calculated fold change/ratio. We thank the three reviewers for their support of our main findings and for the numerous helpful comments aimed at highlighting the context of our discoveries, improving the clarity of the text, and enhancing the potential impact of our results. The revisions introduced in response to their comments and suggestions have greatly improved the clarity of the presentation and the validity of the arguments.

D E P A R T M E N T O F C H E M I C
Below we provide detailed accounts of the responses to the reviewers' comments.

Reviewer 1.
We thank Reviewer 1 for stating that our paper presents "an important contribution to the understanding of inhibition of β-hematin beyond classical mechanism based on presence of drug" and for classifying our results as interesting and with a potential to "open a way to future rational designs."

Concern 1. Beyond the difference found in AFM experiment between PY, MQ or CQ with H-ART, what structural aspects could be dominant in the discrete inhibition of H-ART after drug removal?
Response 1. The mechanism of irreversible inhibition by H-ART initiates with the enhanced nucleation of nanocrystals in the presence of H-ART. The newly nucleated crystallites associate to the surface of larger growing crystals where they incorporate and strain the lattice. The generated lattice strain lowers the crystallization driving force and suppresses crystal growth. The sequence of events that follow enhanced crystal nucleation appears to be general, and are likely to be triggered by any compound that enhances nucleation. This expectation is supported by results with H-ARS. Thus, the question of the uniqueness of the mechanism of irreversible inhibition by H-ART transforms into why does H-ART enhance nucleation. We fully agree with Reviewer 1 that this is an extremely intriguing question on its own right. We are currently finalizing a study of how solution properties and mesoscopic solution aggregates couple to hematin crystal nucleation to address this point. This study will be published separately. The short answer is that H-ART and H-ARS, analogously to hematin, carry carboxyl groups. We use mixed aqueous-organic solvent to mimic the hemozoin environment in the parasite digestive vacuole. In this solvent the carboxyl groups of the heme adducts and hematin partially deprotonate and the resulting Coulomb repulsion between the carboxylate anions boosts the crystallization driving force and hematin crystal nucleation. Inhibitors PY, CQ, and MQ carry amino groups and do not have this effect.

Concern 2.
Have author mediated the possibility to reply the biological studies by using CQ-resistant strain? It can be very interesting in order to explore the occurrence or nor of this non-classical mechanism in CQ-resistant strain of P. falciparum? Response 1. We fully agree with Reviewer 1 that the responses to short drug pulses of a CQ-resistant strain would provide valuable insights. We have ongoing studies, to be published shortly, with the P. falciparum strain CAMWT, which is chloroquine resistant and CAM 580Y, which is isogenic and artemisinin ring-stage resistant. We would like to also refer to the work of Roepe that brought up the perspective that chloroquine resistance by PfCRT altered the continuous IC50 but both were susceptible to high toxic doses of chloroquine. NF54 does not have isogenic CQ resistant isolate which would be better for comparison. Roepe also argues that mutant PfCRT confers nearly all CQ cytostatic resistance as defined by an IC50 shift, but a much smaller component of CQ cytocidal resistance as defined by a lethal dose (LD50) shift. In this study, we are pulsing for short times with higher doses which might impose cytosolic stress in addition to more specific DV heme crystal inhibition by chloroquine.
Concern 3. In survival (%) vs. concentration plots of Figure 5, the units in x-axis are expressed as nm. Should be it mM?
Response 1. We thank Reviewer 1 for noticing that the notation for nanomolar was mistyped. We confirm that nM is correct. The drugs are effective at very lower concentrations.

Reviewer 2.
We thank Reviewer 2 for stating that the topic is quite important and the team of authors are prominent and highly skilled researchers.

Major issues:
Concern 1. The key data are presented in Fig. 1, and distinguishing whether blue and orange bars in Fig. 1e are the same or different in height is essential to interpretation, however, no statistics are given (also bars in extended data Fig. 2). From the error bars (extended Fig. 2 has no error bars at all) it is difficult to ascertain which blue and orange bars are statistically the same vs different. Routine T test and recitation of calculated p values is sorely needed.
Response 1. We thank Reviewer 2 for this valuable suggestion. We have carried out one-way ANOVA (equivalent to the t-test) of the similarities between the distributions of the crystal length and width increments. The ANOVA parameters and the suggested reversibility or irreversibility of inhibition are listed in the updated Supplementary Table 1 and extensively referenced in the text of the revised manuscript.
Concern 2. The authors propose that inhibition of crystal growth is irreversible if two criteria are met, the first being less crystal growth vs control (which is no inhibitor for 13 days) in the 10 days after inhibitor is removed following a 3 day incubation with inhibitor (criterion 1) and, second, that growth in the constant presence of inhibitor for 13 days is the same as growth following 3 days + followed by 10 days -inhibitor. Only 1 inhibitor (H-ARS) seems to satisfy both, but the title and text of the paper seems to imply that the inhibitors are irreversible, and Fig. 2 presents detailed arguments and a cartoon entitled "irreversible inhibition ... by H-ART" when the data in Fig. 1 show that H-ART satisfies neither criterion. The separation of crystal width vs length effects as reversible or irreversible in the text further confuses interpretation, are the authors implying that some inhibitors are reversible in one dimension but irreversible in another ? Some clarification in simple language is needed to assist the reader.
Response 2. We thank Reviewer 2 for pointing out that the discussion of the reversibility of inhibition of bulk crystallization may be confusing. We have rewritten this section of the paper relying on the ANOVA parameters-suggested in Concern 1-and we think that the revised version is substantially clearer. The revised discussion also emphasizes that, owing to the unique molecular structure of each crystal face, inhibitors are not expected to bind equally or to employ an identical mechanism to inhibit {100}, {010} and {011} faces of hematin crystals. We introduce the clarification "Notably the distinct structures of the anisotropic crystal faces select distinct modes of inhibitor binding (to the kinks or on the terraces) and mechanisms and degrees of inhibition on each crystal face. Thus, we do not expect a drug to inhibit all faces uniformly reversibly or irreversibly." Furthermore, we note that H-ART indeed does not inhibit both the {011} faces, which contribute to the crystal length, and the {010} faces, whose growth increases the crystal width; it inhibits irreversibly the {100} faces, as revealed by AFM observations of that face (Fig. 2). To address the concern that an irreversible inhibition of just one face may not have physiological consequences we introduced the following clarification in the revised manuscript: "even if a drug inhibits irreversibly only one of the hematin crystal faces (Fig. 1a), it will still delay the sequestration of hematin and contribute to the accumulation of this product of hemoglobin digestions. Thus, we expect the five compounds tested here to exhibit irreversible suppression of malaria parasites." Concern 3. The crystal inhibition assays are done over 13 days in vitro under highly non physiologic conditions, but the authors imply that by comparing growth effects of live parasites vs the same inhibitors that somehow the measured crystal inhibition characteristics are relevant to understanding the mechanism of drug inhibition of hemozoin in vivo, in which hemozoin crystals are formed within hours. The highly artificial nature of the crystal formation measurements needs to be emphasized, with limitations on interpretation then highlighted.
Response 3. In our in vitro assays we use biomimetic solvents with hematin concentrations similar to those in the parasite DV [Heller LE & Roepe PD (2018) Biochemistry 57(51):6927-6934]. Crystallization trials were carried out for extended times to test whether three distinct regimes of application of drugs and metabolites induced divergent crystal sizes. The comparisons between the average crystal sizes resulting from the three growth regimes indicated that mechanisms of irreversible inhibition of crystallization operate for the studied drugs and metabolites. These mechanisms were than confirmed in AFM experiments, which were carried out over times between one and three hours. Thus, the times of the bulk crystallization and the AFM tests bracket the times of parasite growth. We have modified the language in the revised manuscript to reflect the difference between death in 3 to 6 hours at the stage drug applied versus lingering drug in the digestive vacuole or on hemozoin, which persists to further damage the parasite growth after wash out. The off digestive vacuole target effects on ring stage cytosol toxicity versus trophozoite stage sensitivity may be different as more antioxidant molecules may be present, such as GSH, in the parasite cytosol at the trophozoite stage. We note that pyronaridine is more efficient at the ring stage killing than trophozoite, whereas chloroquine and amodiaquine were more efficient at killing at the trophozoite stage.